Human serum albumin (HSA) is a protein whose mature form consists of 585 amino acids. HSA is the most abundant component of plasma proteins, having a long half-life of 14-21 days in the plasma. HSA contributes to adjustment of osmotic pressure of the plasma, and functions to bind to, and carry, intrinsic compounds such as cations, fatty acids, hormones, bilirubin, and the like as well as extrinsic ones like medicines in the blood. In general, compounds bound to HSA become less likely to be absorbed by organs, and thus can circulate for a longer time in the blood.
Human serum albumin (HSA) is known to have plural natural variants. Human serum albumin Redhill is one of them (Non-patent documents 1 and 2). In comparison with the amino acid sequence of the common human serum albumin consisting of 585 amino acids as mentioned above, human serum albumin Redhill differs in that alanine as the 320th amino acid residue from the N-terminus is replaced with threonine, and that one arginine residue is added to the N-terminus, and it thus consists of 586 amino acids. This replacement of alanine with threonine give rise to a sequence Asn-Tyr-Thr within the amino acid sequence of albumin Redhill, and this Asn (asparagine) residue in that sequence receives N-glycosylation. Thus, the molecular weight of albumin Redhill is observed to be greater than the above common human serum albumin by approximately 2.5 kDa.
There is reported a method to increase the stability of a protein, such as an enzyme, in plasma by fusing HSA with the protein (Non-patent document 3, Patent documents 1 and 2). A fusion protein made of HSA and an enzyme or the like is provided in a medium or within cells as a recombinant protein, by culturing transformant cells produced by introducing an expression vector carrying a DNA in which a gene encoding HSA and a gene encoding a protein, e.g., an enzyme, are linked in frame.
Examples of proteins whose stability in plasma is increased by fusion with human serum albumin (HSA) include a fusion protein of HSA with G-CSF (Patent documents 1 and 3), a fusion protein of HSA with interferon α (Patent document 4), a fusion protein of HSA with GLP-1 (Patent document 5), a fusion protein of HSA with insulin (Patent document 6), a fusion protein of HSA with erythropoietin (Patent document 7), a fusion protein of HSA with growth hormone (Patent documents 4, 5 and 8-11), and the like.
Human growth hormone (hGH) is a protein secreted from the anterior pituitary under the control of hypothalamus. Human GH exhibits growth-promoting activities such as promotion of cartilage formation, promotion of protein anabolism, and the like, as well as improvement of body composition and lipid metabolism. Children with low hGH secretion exhibit growth hormone deficiency dwarfism, which is characterized by low height compared with normal children.
Pharmaceutical preparations (hGH preparation) containing hGH as the active principle, which is prepared as a recombinant protein utilizing E. coli cells with an introduced hGH gene and has molecular weight of approximately 22 kD, are clinically used widely as a therapeutic drug for growth hormone deficiency dwarfism, dwarfism in Turner syndrome, dwarfism in SGA (Small-for-Gestational Age), dwarfism by chronic renal failure, dwarfism in Prader-Willi syndrome, and dwarfism in achondroplasia, accompanied by no epiphyseal closure. After subcutaneous or intramuscular administration of an hGH preparation, it circulates in the blood, and its growth-promoting activity promotes growth of the patient. Preparations containing hGH are clinically used widely also as a therapeutic drug for adult growth hormone deficiency. Patients with adult growth hormone deficiency show various abnormalities such as abnormal lipid metabolism, and administration of hGH preparation will bring about improved QOL of the patients through, e.g., normalization of patients' lipid metabolism. Growject™, e.g., is available as an hGH preparation for growth hormone deficiency dwarfism and adult growth hormone deficiency.
Those attempts to improve stability of hGH in plasma were made in response to clinical needs. The half-life of hGH in plasma is regarded to be less than 20 minutes, and hGH administered to a patient thus quickly disappears from the blood. For hGH to exhibit its pharmacological activity in a patient, therefore, it must be administered to the patient either three times a week intramuscularly or everyday subcutaneously. Such frequent administration imposes a burden on patients. So, reduction of administration frequency, if achieved by increasing the stability of hGH in plasma and thereby elongating its half-life in plasma, would be desirable as leading to reduction of patients' burden.